SUMMARY Central tolerance can be mediated by peripheral dendritic cells (DCs) that transport innocuous antigens (Ags) to the thymus for presentation to developing T cells, but the responsible DC subsets remain poorly defined. Immature plasmacytoid DCs (pDCs) express CCR9, a chemokine receptor involved in migration of T cell precursors to the thymus. We show here that CCR9 mediated efficient thymic entry of endogenous or i.v. transfused pDCs. pDCs activated by Toll-like receptor (TLR) ligands downregulated CCR9 and lost their ability to home to the thymus. Moreover, endogenous pDCs took up subcutaneously injected fluorescent Ag and, in the absence of TLR signals, transported Ag to the thymus in a CCR9-dependent fashion. Injected, Ag-loaded pDCs effectively deleted Ag-specific thymocytes, and this thymic clonal deletion required CCR9-mediated homing and was prevented by infectious signals. Thus peripheral pDCs can contribute to immune tolerance through CCR9-dependent transport of peripheral Ags and subsequent deletion of Ag-reactive thymocytes.

Chemerin recruits NK cells to suppress melanoma growth.

T-cell-based immunotherapies provide a promising means of cancer treatment although durable antitumor responses are infrequent. A potential reason for these shortcomings may lie in the observed lack of trafficking of specific T cells to tumor. Our increasing knowledge of the process of trafficking involving adhesion molecules and chemokines affords us the opportunity to intervene and correct deficiencies in this process. Chemokines can be expressed by a range of tumors and may serve as suitable targets for directing specific T cells toward tumor. We initially sought to identify which chemokines were produced by a range of human tumor cell lines, and which chemokines and chemokine receptors were expressed by cultured T cells. We identified two chemokines: Growth-Regulated Oncogene-alpha (Gro-alpha; CXCL1) and Regulated on Activation Normal T Cell-Expressed and Secreted (RANTES; CCL5), to be secreted by several human tumor cell lines. Expression was also detected in fine-needle aspirates of melanoma from patients. In addition, we determined the expression of several chemokine receptors on cultured human T cells including CCR1, CCR2, CCR4, CCR5, CXCR3, and CXCR4. Cultured, activated human T cells expressed the chemokines lymphotactin (XCL1), RANTES, macrophage inflammatory protein-1 alpha (MIP-1 alpha; CCL3) and MIP-1 beta (CCL4), but no appreciable Gro-alpha. In a strategy to direct T cells toward chemokines expressed by tumors we chose Gro-alpha as the target chemokine because it was produced by tumor and not by T cells themselves. However, T cells did not express the receptor for Gro-alpha, CXCR2, and therefore, T cells were transduced with a retroviral vector encoding CXCR2. Calcium ion mobilization, an important first step in chemokine receptor signaling, was subsequently demonstrated in transduced T cells in response to Gro-alpha. In addition, Gro-alpha was chemotactic for T cells expressing CXCR2 in vitro toward both recombinant protein and tumor-derived chemokine. Interestingly we demonstrate, for the first time, that Gro-alpha was able to induce interferon-gamma (IFN-gamma) secretion from transduced T cells, thereby extending our knowledge of other potential functions of CXCR2. This study demonstrates the feasibility of redirecting the migration properties of T cells toward chemokines secreted by tumors.

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Antibody-dependent cell-mediated cytotoxicity (ADCC), which is largely mediated by natural killer (NK) cells, is thought IntroductionMonoclonal antibodies (mAbs) have revolutionized the treatment of cancer. The first approved mAb for this purpose, rituximab, a murine-human chimeric immunoglobulin G1 (IgG1) antibody against CD20, has become a standard treatment for patients with B-cell lymphomas.Despite tumor response rates to rituximab of up to 90% and decreased risk of death by as much as 36%, the majority of patients with advanced lymphoma still die of their disease, including 19 000 patients in the United States in 2009 alone. [1][2][3][4] Enhancing the efficacy of rituximab represents an opportunity to improve patient outcome. We have developed a strategy to enhance the antitumor activity of rituximab by augmenting antibody-induced cell killing.Several mechanisms of rituximab's antitumor action have been proposed, including direct induction of apoptosis, complementdependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), and, possibly, induction of an adaptive immune response (a "vaccinal" effect). 5 Among these mechanisms, ADCC is believed to be of importance, particularly to the initial antitumor response. In vitro studies have shown that rituximab can induce ADCC of human lymphoma cell lines. 6 In murine xenotransplant lymphoma models, a role for ADCC in rituximab's efficacy was confirmed in studies using FcR-␥-chain-deficient mice, 7 as well as a neutralizing antibody against murine Fc␥R. 8 Further murine studies using CD20 mAbs have confirmed that monocyte-mediated ADCC is the primary, if not exclusive, mechanism through which normal and malignant B cells are depleted in vivo. 9-13 Finally, clinical results have shown that patients harboring an Fc␥RIIIA polymorphism with higher affinity for IgG1 have better responses to rituximab, further supporting the hypothesis that ADCC is an important in vivo mechanism of rituximab action in patients with lymphoma. 14,15 Natural killer (NK) cells are known to be important effector cells mediating ADCC. Binding of the NK-cell Fc receptor (Fc␥RIII, CD16) to the constant region of an antibody induces NK-cell activation. On activation, NK cells release cytotoxic granules, promoting tumor cell killing, and up-regulate the expression of several activation markers, including CD137. 16 In this study, we hypothesized that rituximab-induced ADCC could be specifically increased by using an anti-CD137 agonistic mAb to enhance NK-cell function.CD137 (4-1BB) is a surface glycoprotein that belongs to the tumor-necrosis factor receptor superfamily. 17 CD137 is an inducible costimulatory molecule expressed on a variety of immune cells, including activated CD4 and CD8 T cells, NK cells, monocytes, and dendritic cells. 18,19 On T cells specifically, CD137 functions as a costimulatory receptor induced on T-cell receptor stimulation. In this context, ligation of CD137 leads to increased T-cell proliferation, cytokine production, functional maturation, and prolonged ...

Non-Hodgkin lymphoma (NHL) presents as both localized and disseminated disease with spread to secondary sites carrying a worse prognosis. Although pathways driving NHL dissemination have been identified, there are few therapies capable of inhibiting them. Here, we report a novel role for the immunomodulatory protein CD47 in NHL dissemination, and we demonstrate that therapeutic targeting of CD47 can prevent such spread. We developed 2 in vivo lymphoma metastasis models using Raji cells, a human NHL cell line, and primary cells from a lymphoma patient. CD47 expression was required for Raji cell dissemination to the liver in mouse xenotransplants. Targeting of CD47 with a blocking antibody inhibited Raji cell dissemination to major organs, including the central nervous system, and inhibited hematogenous dissemination of primary lymphoma cells. We hypothesized that anti-CD47 antibodymediated elimination of circulating tumor cells occurred through phagocytosis, a previously described mechanism for blocking anti-CD47 antibodies. As predicted, inhibition of dissemination by anti-CD47 antibodies was dependent on blockade of phagocyte SIRP␣ and required macrophage effector cells. These results demonstrate that CD47 is required for NHL dissemination, which can be therapeutically targeted with a blocking anti-CD47 antibody. Ultimately, these findings are potentially applicable to the dissemination and metastasis of other solid tumors. (Blood. 2011;118(18):4890-4901) IntroductionLymphocyte trafficking is essential for the regulation of systemic immune processes, as well as lymphocyte differentiation and development. Most mature lymphocytes recirculate continuously from blood to tissue and back to the blood again. 1 This recirculation is not random but rather is guided by lymphocyte-endothelial interactions mediated by adhesion molecules (L-selectin, CD44, integrin ␣ 4 ␤ 7 , VLA-4, and LFA-1) and select chemokines. 2,3 Malignant transformation of normal lymphocytes results in lymphoma, many subtypes of which migrate and disseminate. Unlike the metastasis of other cancers, lymphoma dissemination probably reflects conserved physiologic behavior, rather than acquisition of metastatic potential. Indeed, mechanisms of normal lymphocyte homing and recirculation have been implicated in lymphoma dissemination and invasion. For example, adhesion molecules involved in normal lymphocyte trafficking have been shown to play a role in lymphoma dissemination, including LFA-1, ␣ 3 ␤ 3 , and other homing-associated integrins. [4][5][6] Furthermore, several of these adhesion molecules have been therapeutically exploited, as antibodies targeting the adhesion receptors LFA-1, integrin ␣ v ␤ 3 , and CD44 can inhibit dissemination of lymphoma in experimental models. 5,[7][8][9] CD47, also known as integrin-associated protein, has been implicated in the migration and mobilization of normal leukocytes. [10][11][12][13][14] In cancer, we recently demonstrated that CD47 regulates lymphoma pathogenesis by enabling evasion of phagocytosis through bind...

Immunotherapy is an important breakthrough in cancer. US Food and Drug Administration-approved immunotherapies for cancer treatment (including, but not limited to, sipuleucel-T, ipilimumab, nivolumab, pembrolizumab, and atezolizumab) substantially improve overall survival across multiple malignancies. One mechanism of action of these treatments is to induce an immune response against antigen-bearing tumor cells; the resultant cell death releases secondary (nontargeted) tumor antigens. Secondary antigens prime subsequent immune responses (antigen spread). Immunotherapy-induced antigen spread has been shown in clinical studies. For example, in metastatic castration-resistant prostate cancer patients, sipuleucel-T induced early immune responses to the immunizing antigen (PA2024) and/or the target antigen (prostatic acid phosphatase). Thereafter, most patients developed increased antibody responses to numerous secondary proteins, several of which are expressed in prostate cancer with functional relevance in cancer. The ipilimumab-induced antibody profile in melanoma patients shows that antigen spread also occurs with immune checkpoint blockade. In contrast to chemotherapy, immunotherapy often does not result in short-term changes in conventional disease progression end points (eg, progression-free survival, tumor size), which may be explained, in part, by the time taken for antigen spread to occur. Thus, immune-related response criteria need to be identified to better monitor the effectiveness of immunotherapy. As immunotherapy antitumor effects take time to evolve, immunotherapy in patients with less advanced cancer may have greater clinical benefit vs those with more advanced disease. This concept is supported by prostate cancer clinical studies with sipuleucel-T, PSA-TRICOM, and ipilimumab. We discuss antigen spread with cancer immunotherapy and its implications for clinical outcomes.

Contributors NMC and LCH were responsible for study design, study coordination, cumulative data collection, data interpretation, and primary manuscript writing. WX did the statistical analyses and participated in the manuscript drafting.